Glp-1 analogues

ABSTRACT

The present invention is directed to peptide analogues of glucagon-like peptide-1, the pharmaceutically-acceptable salts thereof, to methods of using such analogues to treat mammals and to pharmaceutical compositions useful therefor comprising said analogues.

BACKGROUND OF THE INVENTION

The present invention is directed to peptide analogues of glucagon-likepeptide-1, the pharmaceutically-acceptable salts thereof, to methods ofusing such analogues to treat mammals and to pharmaceutical compositionsuseful therefor comprising said analogues.

Glucagon-like peptide-1 (7-36) amide (GLP-1) (SEQ ID NO: 1) issynthesized in the intestinal L-cells by tissue-specificpost-translational processing of the glucagon precursor pre-proglucagon(Varndell, J. M., et al., J. Histochem Cytochem, 1985:33:1080-6) and isreleased into the circulation in response to a meal. The plasmaconcentration of GLP-1 rises from a fasting level of approximately 15pmol/L to a peak postprandial level of 40 pmol/L. It has beendemonstrated that, for a given rise in plasma glucose concentration, theincrease in plasma insulin is approximately threefold greater whenglucose is administered orally compared with intravenously (Kreymann,B., et al., Lancet 1987:2, 1300-4). This alimentary enhancement ofinsulin release, known as the incretin effect, is primarily humoral andGLP-1 is now thought to be the most potent physiological incretin inhumans. In addition to the insulinotropic effect, GLP-1 suppressesglucagon secretion, delays gastric emptying (Wettergren A., et al., DigDis Sci 1993:38:665-73) and may enhance peripheral glucose disposal(D'Alessio, D. A. et al., J. Clin Invest 1994:93:2293-6).

In 1994, the therapeutic potential of GLP-1 was suggested following theobservation that a single subcutaneous (s/c) dose of GLP-1 couldcompletely normalize postprandial glucose levels in patients withnon-insulin-dependent diabetes mellitus (NIDDM) (Gutniak, M. K., et al.,Diabetes Care 1994:17:1039-44). This effect was thought to be mediatedboth by increased insulin release and by a reduction in glucagonsecretion. Furthermore, an intravenous infusion of GLP-1 has been shownto delay postprandial gastric emptying in patients with NIDDM (Williams,B., et al., J. Clin Endo Metab 1996:81:327-32). Unlike sulfonylureas,the insulinotropic action of GLP-1 is dependent on plasma glucoseconcentration (Holz, G. G. 4^(th), et al., Nature 1993:361:362-5). Thus,the loss of GLP-1-mediated insulin release at low plasma glucoseconcentration protects against severe hypoglycemia. This combination ofactions gives GLP-1 unique potential therapeutic advantages over otheragents currently used to treat NIDDM.

Numerous studies have shown that when given to healthy subjects, GLP-1potently influences glycemic levels as well as insulin and glucagonconcentrations (Orskov, C, Diabetologia 35:701-711, 1992; Holst, J. J.,et al., Potential of GLP-1 in diabetes management in Glucagon III,Handbook of Experimental Pharmacology, Lefevbre P J, Ed. Berlin,Springer Verlag, 1996, p. 311-326), effects which are glucose dependent(Kreymann, B., et al., Lancet ii: 1300-1304, 1987; Weir, G. C., et al.,Diabetes 38:338-342, 1989). Moreover, it is also effective in patientswith diabetes (Gutniak, M., N. Engl J Med 226:1316-1322, 1992; Nathan,D. M., et al., Diabetes Care 15:270-276, 1992), normalizing bloodglucose levels in type 2 diabetic subjects (Nauck, M. A., et al.,Diagbetologia 36:741-744, 1993), and improving glycemic control in type1 patients (Creutzfeldt, W. O., et al., Diabetes Care 19:580-586, 1996),raising the possibility of its use as a therapeutic agent.

GLP-1 is, however, metabolically unstable, having a plasma half-life(t_(1/2)) of only 1-2 min in vivo. Exogonously administered GLP-1 isalso rapidly degraded (Deacon, C. F., et al., Diabetes 44:1126-1131,1995). This metabolic instability limits the therapeutic potential ofnative GLP-1. Hence, there is a need for GLP-1 analogues that are moreactive or are more metabolically stable than native GLP-1.

SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to a compound offormula (I),

(R²R³)-A⁷-A⁸-A⁹-A¹⁰-A¹¹-A¹²-A¹³-A¹⁴-A¹⁵-A¹⁶-A¹⁷-A¹⁸-A¹⁹-A²⁰-A²¹-A²²-A²³-A²⁴-A²⁵-A²⁶-A²⁷-A²⁸-A²⁹-A³⁰-A³¹-A³²-A³³-A³⁴-A³⁵-A³⁶-A³⁷-R¹,  (I)

whereinA⁷ is L-His, Ura, Paa, Pta, D-His, Tyr, 3-Pal, 4-Pal, Hppa, Tma-His, Ampor deleted, provided that when A⁷ is Ura, Paa, Pta or Hppa then R² andR³ are deleted;

A⁸ is Ala, D-Ala, Aib, Acc, N-Me-Ala, N-Me-D-Ala, Arg or N-Me-Gly; A⁹ isGlu, N-Me-Glu, N-Me-Asp or Asp; A¹⁰ is Gly, Acc, Ala, D-Ala, Phe or Aib;A¹¹ is Thr or Ser; A¹² is Phe, Acc, Aic, Aib, 3-Pal, 4-Pal, β-Nal, Cha,Trp or X¹-Phe; A¹³ is Thr or Ser; A¹⁴ is Ser, Thr, Ala or Aib; A¹⁵ isAsp, Ala, D-Asp or Glu; A¹⁶ is Val, D-Val, Acc, Aib, Leu, Ile, Tle, Nle,Abu, Ala, D-Ala, Tba or Cha; A¹⁷ is Ser, Ala, D-Ala, Aib, Acc or Thr;A¹⁸ is Ser, Ala, D-Ala, Aib, Acc or Thr; A¹⁹ is Tyr, D-Tyr, Cha, Phe,3-Pal, 4-Pal, Acc, β-Nal, Amp or X¹-Phe; A²⁰ is Leu, Ala, Acc, Aib, Nle,Ile, Cha, Tle, Val, Phe or X¹-Phe; A²¹ is Glu, Ala or Asp; A²² is Gly,Acc, Ala, D-Ala, β-Ala or Aib; A²³ is Gln, Asp, Ala, D-Ala, Aib, Acc,Asn or Glu; A²⁴ is Ala, Aib, Val, Abu, Tle or Acc;

A²⁵ is Ala, Aib, Val, Abu, Tle, Acc, Lys, Arg, hArg, Orn, HN—CH((CH₂),—NR¹⁰R¹¹)—C(O) or HN—CH((CH₂)_(e)—X³)—C(O);A²⁶ is Lys, Ala, 3-Pal, 4-Pal, Arg, hArg, Orn, Amp,HN—CH((CH₂)_(n)—NR¹⁰R¹¹)—C(O) or HN—CH((CH₂)_(e)—X³)—C(O);

A²⁷ is Glu, Ala, D-Ala or Asp; A²³ is Phe, Ala, Pal, β-Nal, X¹-Phe, Aic,Acc, Aib, Cha or Trp; A²⁹ is Ile, Acc, Aib, Leu, Nle, Cha, Tle, Val,Abu, Ala, Tba or Phe;

A³⁰ is Ala, Aib, Acc or deleted;A³¹ is Trp, Ala, β-Nal, 3-Pal, 4-Pal, Phe, Acc, Aib, Cha, Amp ordeleted;A³² is Leu, Ala, Acc, Aib, Nle, Ile, Cha, Tle, Phe, X¹-Phe, Ala ordeleted;A³³ is Val, Acc, Aib, Leu, Ile, Tle, Nle, Cha, Ala, Phe, Abu, X¹-Phe,Tba, Gaba or deleted;A³⁴ is Lys, Arg, hArg, Orn, Amp, Gaba, HN—CH((CH₂), —NR¹⁰R¹¹)—C(O),HN—CH((CH₂)_(e)—X³)—C(O) or deleted;A³⁵ is Gly or deleted;A³⁶ is L- or D-Arg, D- or L-Lys, D- or L-hArg, D- or L-Orn, Amp,HN—CH((CH₂), —NR¹⁰R¹¹)—C(O), HN—CH((CH₂)_(e)—X³)—C(O) or deleted;A³⁷ is Gly or deleted;

-   -   X¹ for each occurrence is independently selected from the group        consisting of (C₁-C₆)alkyl, OH and halo;    -   R¹ is OH, NH₂, (C₁-C₁₂)alkoxy, or NH—X²—CH₂-Z⁰, wherein X² is a        (C₁-C₁₂)hydrocarbon moiety, and Z⁰ is H, OH, CO₂H or CONH₂;    -   X³ is

or —C(O)—NHR¹², wherein X⁴ for each occurrence is independently —C(O)—,—NH—C(O)— or —CH₂—, and f for each occurrence is independently aninteger from 1 to 29;

-   -   each of R² and R³ is independently selected from the group        consisting of H, (C₁-C₃₀)alkyl, (C₂-C₃₀)alkenyl,        phenyl(C₁-C₃₀)alkyl, naphthyl(C₁-C₃₀)alkyl,        hydroxy(C₁-C₃₀)alkyl, hydroxy(C₂-C₃₀)alkenyl,        hydroxyphenyl(C₁-C₃₀)alkyl, and hydroxynaphthyl(C₁-C₃₀)alkyl; or        one of R² and R³ is C(O)X⁵ in which X is (C₁-C₃₀)alkyl,        (C₂-C₃₀)alkenyl, phenyl(C₁-C₃₀)alkyl, naphthyl(C₁-C₃₀)alkyl,        hydroxy(C₁-C₃₀)alkyl, hydroxy(C₂-C₃₀)alkenyl,        hydroxyphenyl(C₁-C₃₀)alkyl, hydroxynaphthyl(C₁-C₃₀)alkyl,

-   -   where Y is H or OH, r is 0-4 and q is 0-4;    -   n for each occurrence is independently an integer from 1-5; and    -   R¹⁰ and R¹¹ for each occurrence is each independently H,        (C₁-C₃₀)alkyl, (C₁-C₃₀)acyl, (C₁-C₃₀)alkylsulfonyl,        —C((NH)(NH₂)) or

provided that when R¹⁰ is (C₁-C₃₀)acyl, (C₁-C₃₀)alkylsulfonyl,—C((NH)(NH₂)) or

-   -   R¹¹ is H or (C₁-C₃₀)alkyl; and    -   R¹² is (C₁-C₃₀)alkyl;        with the proviso that:        (i) at least one amino acid of a compound of formula (I) is not        the same as the native sequence of hGLP-1(7-36, or -37)NH₂ (SEQ        ID NOS: 1, 2) or hGLP-1(7-36, or -37)OH (SEQ ID NOS: 3, 4);        (ii) a compound of formula (I) is not an analogue of        hGLP-1(7-36, or -37)NH₂ (SEQ ID NOS: 1, 2) or hGLP-1(7-36, or        -37)OH (SEQ ID NOS: 3, 4) wherein a single position has been        substituted by Ala;        (iii) a compound of formula (I) is not        [Lys²⁶(N^(ε)-alkanoyl)]hGLP-1(7-36, or -37)-E (SEQ ID NOS: 5-8),        [Lys³⁴(N^(ε)-alkanoyl)]hGLP-1(7-36, or -37)-E (SEQ ID NOS:        9-12), [Lys^(26,34)-bis(N^(ε)-alkanoyl)]hGLP-1(7-36, or -37)-E        (SEQ ID NOS: 13-16), [Arg²⁶, Lys³⁴(N^(ε)-alkanoyl)]hGLP-1(8-36,        or -37)-E (SEQ ID NOS: 17-20), or [Arg^(26,34),        Lys³⁶(N^(ε)-alkanoyl)]hGLP-1(7-36, or -37)-E, wherein E is —OH        or —NH₂ (SEQ ID NOS: 21-24);        (iv) a compound of formula (I) is not Z¹-hGLP-1(7-36, or        -37)-OH, Z¹-hGLP-1(7-36, or -37)-NH₂, where Z¹ is selected from        the group consisting of

-   (a) [Arg²⁶] (SEQ ID NOS: 25-28), [Arg³⁴] (SEQ ID NOS: 29-32),    [Arg^(23,34)] (SEQ ID NOS: 33-36), [Lys³⁶] (SEQ ID NOS: 37-40),    [Arg²⁶, Lys³⁶] (SEQ ID NOS: 41-44), [Arg³, Lys³⁶] (SEQ ID NOS:    45-48), [D-Lys³⁶], [Arg³⁶] (SEQ ID NOS: 3,4,1,2), [D-Arg³⁶],    [Arg^(26,34), Lys³⁶] (SEQ ID NOS: 49-52), or [Arg^(26,36), Lys³⁴]    (SEQ ID NOS: 25-28);

-   (b) [Asp²¹] (SEQ ID NOS: 53-56);

-   (c) at least one of [Aib³] (SEQ ID NOS: 57-60), [D-Ala⁸] and [Asp⁹]    (SEQ ID NOS: 61-64); and

-   (d) [Tyr⁷] (SEQ ID NOS: 65-68), [N-acyl-His⁷] (SEQ ID NOS: 69-72),    [N-alkyl-His⁷], [N-acyl-D-His⁷] (SEQ ID NOS: 73-76) or    [N-alkyl-D-His⁷];    (v) a compound of formula (I) is not a combination of any two of the    substitutions listed in groups (a) to (d); and    (vi) a compound of formula (I) is not [N-Me-Ala⁸]hGLP-1(8-36 or -37)    (SEQ ID NOS: 75, 78), [Glu¹]hGLP-1(7-36 or -37) (SEQ ID NOS: 79,    80), [Asp²¹]hGLP-1(7-36 or -37) (SEQ ID NOS: 53, 54) or    [Phe³¹]hGLP-1(7-36 or -37) (SEQ ID NOS: 81, 82).

A preferred compound of the immediately foregoing compound of formula(I) is where A¹¹ is Thr; A¹³ is Thr; A¹⁴ is Ser, Aib or Ala; A¹⁷ is Ser,Ala, Aib or D-Ala; A¹⁸ is Ser, Ala, Aib or D-Ala; A²¹ is Glu or Ala; A²³is Gln, Glu, or Ala; and A²⁷ is Glu or Ala; or a pharmaceuticallyacceptable salt thereof.

A preferred compound of the immediately foregoing compound of formula(I) is where A⁹ is Glu, N-Me-Glu or N-Me-Asp; A¹² is Phe, Acc or Aic;A¹⁶ is Val, D-Val, Acc, Aib, Ala, Tle or D-Ala; A¹⁹ is Tyr, 3-Pal, 4-Palor D-Tyr; A²⁰ is Leu, Acc, Cha, Ala or Tle; A²⁴ is Ala, Aib or Acc; A²⁵is Ala, Aib, Acc, Lys, Arg, hArg, Orn, HN—CH((CH₂), —NH—R¹⁰)—C(O); A²⁸is Phe or Ala; A²⁹ is Ile, Acc or Tle; A³⁰ is Ala, Aib or deleted; A³¹is Trp, Ala, 3-Pal, 4-Pal or deleted; A³² is Leu, Acc, Cha, Ala ordeleted; A³³ is Val, Acc, Ala, Gaba, Tle or deleted; or apharmaceutically acceptable salt thereof.

A preferred compound of the immediately foregoing compound of formula(I) is where A⁸ is Ala, D-Ala, Aib, A6c, A5c, N-Me-Ala, N-Me-D-Ala orN-Me-Gly; A¹⁰ is Gly, Ala, D-Ala or Phe; A¹² is Phe, A6c or A5c; A¹⁶ isVal, Ala, Tle, A6c, A5c or D-Val; A²⁰ is Leu, A6c, A5c, Cha, Ala or Tle;A²² is Gly, Aib, β-Ala, L-Ala or D-Ala; A²⁴ is Ala or Aib; A²⁹ is Ile,A6c, A5c or Tle; A³² is Leu, A6c, A5c, Cha, Ala or deleted; A³³ is Val,A6c, A5c, Ala, Gaba, Tle or deleted; or a pharmaceutically acceptablesalt thereof.

A preferred compound of the immediately foregoing compound of formula(I) is where R¹ is OH or NH₂ or a pharmaceutically acceptable saltthereof.

A preferred compound of the immediately foregoing compound of formula(I) or a pharmaceutically acceptable salt thereof is where R² is H andR³ is (C₁-C₃₀)alkyl, (C₂-C₃₀)alkenyl, (C₁-C₃₀)acyl,

A most preferred compound of formula (I) is where said compound is[D-Ala⁸, Ala^(17,23,27), 3-Pal^(19,31), Gaba³⁴]-GLP-1(7-34)NH₂;[D-Ala^(8,23,27), 3-Pal^(19,31)]hGLP-1(7-35)-NH₂; [Ala^(18,23,27),3-Pal^(19,31)]hGLP-1(7-35)-NH₂ (SEQ ID NO: 83); [Ala^(16,23,27),3-Pal^(19,31)]hGLP-1(7-35)-NH₂ (SEQ ID NO: 84); [Ala^(14,23,27)3-Pal^(19,31)]hGLP-1(7-35)-NH₂(SEQ ID NO: 85); [Ala^(22,23,27),3-Pal^(19,31)]hGLP-1(7-35)-NH₂ (SEQ ID NO: 86); [Hppa⁷]hGLP-1(7-36)-NH₂(SEQ ID NO: 87); [Ala^(23,27), 3-Pal^(19,31)]hGLP-1(7-35)-NH₂ (SEQ IDNO: 88); [Ala^(7,23,27), 3-Pal^(19,31)]hGLP-1(7-35)-NH₂ (SEQ ID NO: 89);[Ala^(22,23,27), 3-Pal^(19,31), Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 90);[Ala^(15,22,23,27), 3-Pal^(19,31), Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO:91); [Ala^(17,22,23,27), 3-Pal^(19,31), Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ IDNO: 92); [Ala^(8,22,23,27), 3-Pal^(19,31), Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQID NO: 93); [Ala^(21,22,23,27), 3-Pal^(19,31), Gaba³⁴]hGLP-1(7-34)-NH₂(SEQ ID NO: 94); [Ala^(22,23,26,27), 3-Pal^(19,31),Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 95); [Ala^(22,23,27,32),3-Pal^(19,31), Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 96);[Ala^(22,23,26,27), 3-Pal^(19,31), Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO:97); [Ala^(22,23,27,31), 3-Pal¹⁹, Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO:98); [Ala^(22,23,27,28), 3-Pal^(19,31), Gaba³³]hGLP-1(7-33)-NH₂ (SEQ IDNO: 99); [Ala^(22,23,27,29), 3-Pal^(19,31), Gaba³³]hGLP-1(7-33)-NH₂ (SEQID NO: 100); [Ala^(23,27), 3-Pal^(19,31), Gaba³³]hGLP-1(7-33)-NH₂ (SEQID NO: 101); [Ala^(20,22,23,27), 3-Pal^(19,31), Gaba³³]hGLP-1(7-33)-NH₂(SEQ ID NO: 102); [Ala^(22,23,27), 3-Pal^(19,31),Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 103); [Ala^(17,22,23,27),3-Pal^(19,31), Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 104); [D-Ala¹⁰,Ala^(22,23,27), 3-Pal^(19,31), Gaba³³]hGLP-1(7-33)-NH₂; [D-Ala⁸,Ala^(17,23,27), 3-Pal^(19,31)]hGLP-1(7-34)-NH₂; [Ala^(17,23,27),3-Pal^(19,26,31)]hGLP-1(7-34)-NH₂ (SEQ ID NO: 105); [D-Ala⁸, Ala¹⁷,3-Pal^(19,31)]hGLP-1(7-34)-NH₂; [Ala^(17,23,27),3-Pal^(19,31)]hGLP-1(7-34)-NH₂ (SEQ ID NO: 106); [D-Ala⁸,Ala^(17,23,27), 3-Pal³¹, Tle²⁹]hGLP-1(7-34)-NH₂; [D-Ala⁸,Ala^(17,23,27), 3-Pal^(19,31), Tle¹⁶]hGLP-1(7-34)-NH₂; [D-Ala⁸,Ala^(17,23,27), 3-Pal^(19,31), Gaba³⁴]hGLP (7-34)-NH₂; [D-Ala²²,Ala^(17,23,27), 3-Pal^(19,31), Gaba³⁴]hGLP-1(7-34)-NH₂; [Aib⁸,Ala^(17,23,27), 3-Pal^(19,31), Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 107);[D-Ala⁸, Ala^(17,22,23,27), 3-Pal^(19,31]hGLP-)1(7-33)-NH₂; [Aib⁸,Ala^(17,22,23,27), 3-Pal¹⁹³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 108);[Ala^(17,18,23,27), 3-Pal^(9,3), Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO:109); [Ala^(17,23,27), 3-Pal^(19,31), Tle³³, Gaba³⁴]hGLP-1(7-34)-NH₂(SEQ ID NO: 110); [Tle¹⁶, Ala^(17,23,27), 3-Pal^(19,31),Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 111); [N-Me-D-Ala⁸,Ala^(17,22,23,27), 3-Pal^(19,31)]hGLP-1(7-33)-NH₂; [Aib⁸,Ala^(17,22,23,27), 3-Pal^(19,31)]hGLP-1(7-33)-NH₂ (SEQ ID NO: 112);[Ala^(17,18,22,23,27), 3-Pal^(19,31), Tle^(16,20),Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 113); [D-Ala⁶, Ala^(17,18,22,23,27),3-Pal^(19,31), Tle¹⁶, Gaba³⁴]hGLP-1(7-34)-NH₂; [D-Ala^(8,22)Ala^(17,18,23,27), 3-Pal^(19,31), Gaba³⁴]hGLP-1(7-34)-NH₂;[D-Ala^(8,18), Ala^(17,22,23,27), 3-Pal^(19,31),Gaba³⁴]hGLP-1(7-34)-NH₂; [D-Ala^(8,17), Ala^(18,22,23,27),3-Pal^(19,31), Gaba³⁴]hGLP-1(73)-NH₂; [Ala^(17,18,22,23,27),3-Pal^(19,31), Gaba³⁴]hGLP-1(7-34)-NH₂; or a pharmaceutically acceptablesalt thereof.

Another most preferred compound of formula (I) is wherein said compoundis

[Aib⁸, A6c³²]hGLP-1(7-36)NH₂ (SEQ ID NO: 114);[A6c^(20,32)]hGLP-1(7-36)-NH₂ (SEQ ID NO: 115); [Aib⁹]hGLP-1(7-36)-NH₂(SEQ ID NO: 116); [(Tma-His)⁷]hGLP-1(7-36)-NH₂ (SEQ ID NO: 117);[A6c⁸]hGLP-1(8-36)-NH₂ (SEQ ID NO:118); [A6c⁸]hGLP-1(7-36)-NH₂ (SEQ IDNO: 119); [A6c^(16,20)]hGLP-1(7-36)-NH₂ (SEQ ID NO: 120);[A6c^(29,32)]hGLP-1(7-36)-NH₂ (SEQ ID NO: 121); [A6c²⁰,Aib²⁴]hGLP-1(7-36)-NH₂ (SEQ ID NO: 122); [Aib²⁴,A6c^(29,32)]hGLP-1(7-36)-NH₂ (SEQ ID NO: 123);[A6c^(16,29,32)]hGLP-1(7-36)-NH₂ (SEQ ID NO: 124);[Ura⁷]hGLP-1(7-36)-NH₂ (SEQ ID NO: 125); [Paa⁷]hGLP-1(7-36)-NH₂ (SEQ IDNO: 126); [Pta⁷]hGLP-1(7-36)-NH₂ (SEQ ID NO: 127);[N-Me-Ala⁸]hGLP-1(7-36)-NH₂ (SEQ ID NO: 128);[N-Me-Ala⁸]hGLP-1(8-36)-NH₂; (SEQ ID NO.) [N-Me-D-Ala⁸]hGLP-1(7-36)-NH₂;[N-Me-D-Ala⁸]hGLP-1(8-36)-NH₂; [N-Me-Gly⁸]hGLP-1(7-36)-NH₂ (SEQ ID NO:129); [A5c⁸]hGLP-1(7-36)-NH₂ (SEQ ID NO: 130);[N-Me-Glu⁹]hGLP-1(7-36)-NH₂ (SEQ ID NO: 131); [A5c⁸,A6c^(20,32)]hGLP-1(7-36)-NH₂ (SEQ ID NO: 132); [Aib⁸,A6c³²]hGLP-1(7-36)-NH₂ (SEQ ID NO: 133); [Aib^(8,25)]hGLP-1(7-36)-NH₂(SEQ ID NO: 134); [Aib^(8,24)]hGLP-1(7-36)-NH₂ (SEQ ID NO: 135);[Aib^(8,30)]hGLP-1(7-36)-NH₂ (SEQ ID NO: 136); [Aib⁸,Cha²⁰]hGLP-1(7-36)-NH₂ (SEQ ID NO: 137); [Aib⁸, Cha³²]hGLP-1(7-36)-NH₂(SEQ ID NO: 138); [Aib⁸, Glu²³]hGLP-1(7-36)-NH₂ (SEQ ID NO: 139); [Aib⁸,A6c²⁰]hGLP-1(7-36)-NH₂ (SEQ ID NO: 140); [Aib⁸,A6c^(20,32)]hGLP-1(7-36)-NH₂ (SEQ ID NO: 141);[Aib^(8,22)]hGLP-1(7-36)-NH₂ (SEQ ID NO: 142); [Aib⁸,β-Ala²²]hGLP-1(7-36)-NH₂ (SEQ ID NO: 143); [Aib⁸, Lys²⁵]hGLP-1(7-36)-NH₂(SEQ ID NO: 144); [Aib⁸, A6c¹²]hGLP-1(7-36)-NH₂ (SEQ ID NO: 145); [Aib⁸,A6c²⁹]hGLP-1(7-36)-NH₂ (SEQ ID NO: 146); [Aib⁸, A6c³³]hGLP-1(7-36)-NH₂(SEQ ID NO: 147); [Aib^(8,14)]hGLP-1(7-36)NH₂ (SEQ ID NO: 148);[Aib^(8,18)]hGLP-1(7-36)NH₂ (SEQ ID NO: 149); or[Aib^(8,17)]hGLP-1(7-36)NH₂ (SEQ ID NO: 150); or a pharmaceuticallyacceptable salt thereof. In another aspect, the present inventionprovides a pharmaceutical composition comprising an effective amount ofa compound of formula (I) as defined hereinabove or a pharmaceuticallyacceptable salt thereof and a pharmaceutically acceptable carrier ordiluent.

In still another aspect, the present invention provides a method ofeliciting an agonist effect from a GLP-1 receptor in a subject in needthereof which comprises administering to said subject an effectiveamount of a compound of formula (I) as defined hereinabove or apharmaceutically acceptable salt thereof.

In yet a further aspect, this invention provides a method of treating adisease selected from the group consisting of Type I diabetes, Type IIdiabetes, obesity, glucagonomas, secretory disorders of the airway,metabolic disorder, arthritis, osteoporosis, central nervous systemdisease, restenosis and neurodegenerative disease, renal failure,congestive heart failure, nephrotic syndrome, cirrhosis, pulmonaryedema, hypertension, and disorders wherein the reduction of food intakeis desired, in a subject in need thereof which comprises administeringto said subject an effective amount of a compound of formula (I) asdefined hereinabove or a pharmaceutically acceptable salt thereof.Preferred of the foregoing method is where the disease is Type Idiabetes or Type II diabetes.

With the exception of the N-terminal amino acid, all abbreviations (e.g.Ala) of amino acids in this disclosure stand for the structure of—NH—CH(R)—CO—, wherein R is the side chain of an amino acid (e.g., CH₃for Ala). For the N-terminal amino acid, the abbreviation stands for thestructure of (R²R³)—N—CH(R)—CO—, wherein R is a side chain of an aminoacid and R² and R³ are as defined above except in the case where A⁷ isUra, Paa, Pta or Hppa in which case R² and R³ are not present since Ura,Paa, Pta and Hppa are considered here as des-amino amino acids. Theabbreviations: β-Nal, Nle, Cha, Amp, 3-Pal, 4-Pal and Aib stand for thefollowing α-amino acids: β-(2-naphthyl)alanine, norleucine,cyclohexylalanine, 4-amino-phenylalanine, β-(3-pyridinyl)alanine,β-(4-pyridinyl)alanine and α-aminoisobutyric acid, respectively. Otheramino acid definitions are: Ura is urocanic acid; Pta is (4-pyridylthio)acetic acid; Paa is trans-3-(3-pyridyl)acrylic acid; Tma-His isN,N-tetramethylamidino-histidine; N-Me-Ala is N-methyl-alanine; N-Me-Glyis N-methyl-glycine; N-Me-Glu is N-methyl-glutamic acid; Tle istert-butylglycine; Abu is α-aminobutyric acid; Tba is tert-butylalanine;Orn is ornithine; Aib is α-aminoisobutyric acid; β-Ala is β-alanine;Gaba is γ-aminobutyric acid; Ava is 5-aminovaleric acid; and Aic is2-aminoindane-2-carboxylic acid.

What is meant by Acc is an amino acid selected from the group of1-amino-1-cyclopropanecarboxylic acid (A3c);1-amino-1-cyclobutanecarboxylic acid (A4c);1-amino-1-cyclopentanecarboxylic acid (A5c);1-amino-1-cyclohexanecarboxylic acid (A6c);1-amino-1-cycloheptanecarboxylic acid (A7c);1-amino-1-cyclooctanecarboxylic acid (A8c); and1-amino-1-cyclononanecarboxylic acid (A9c). In the above formula,hydroxyalkyl, hydroxyphenylalkyl, and hydroxynaphthylalkyl may contain1-4 hydroxy substituents. COX⁵ stands for —C═O.X⁵. Examples of —C═O.X⁵include, but are not limited to, acetyl and phenylpropionyl.

What is meant by Lys(N^(ε)-alkanoyl) is represented by the followingstructure:

What is meant by Lys(N^(ε)-alkylsulfonyl) is represented by thefollowing structure:

What is meant by Lys(N^(ε)-(2-(4-alkyl-1-piperazine)-acetyl)) isrepresented by the following structure:

What is meant by Asp(1-(4-alkyl-piperazine)) is represented by thefollowing structure:

What is meant by Asp(1-alkylamino) is represented by the followingstructure:

The variable n in the foregoing structures is 1 to 30.

The full names for other abbreviations used herein are as follows: Bocfor t-butyloxycarbonyl, HF for hydrogen fluoride, Fm for formyl, Xan forxanthyl, Bzl for benzyl, Tos for tosyl, DNP for 2,4-dinitrophenyl, DMFfor dimethylformamide, DCM for dichloromethane, HBTU for2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate, DIEA for diisopropylethylamine, HOAc for aceticacid, TFA for trifluoroacetic acid, 2CIZ for 2-chlorobenzyloxycarbonyland OcHex for Q-cyclohexyl.

A peptide of this invention is also denoted herein by another format,e.g., [A5c⁸]hGLP-1(7-36)-NH₂ (SEQ ID NO: 130), with the substitutedamino acids from the natural sequence placed between the set of brackets(e.g., A5c⁸ for Ala⁸ in hGLP-1). The abbreviation GLP-1 meansglucagon-like peptide-1, and hGLP-1 means human glucagon-like peptide-1.The numbers between the parentheses refer to the number of amino acidspresent in the peptide (e.g., hGLP-1(7-36) (SEQ ID NO: 3) is amino acids7 through 36 of the peptide sequence for human GLP-1). The sequence forhGLP-1(7-37) (SEQ ID NO: 4) is listed in Mojsov, S., Int. J. PeptideProtein Res., 40, 1992, pp. 333-342. The designation “NH₂” inhGLP-1(7-36)NH₂ (SEQ ID NO: 1) indicates that the C-terminus of thepeptide is amidated. hGLP-1(7-36) (SEQ ID NO: 2) means that theC-terminus is the free acid.

DETAILED DESCRIPTION

The peptides of this invention can be prepared by standard solid phasepeptide synthesis. See, e.g., Stewart, J. M., et al., Solid PhaseSynthesis (Pierce Chemical Co., 2d ed. 1984). The substituents R² and R³of the above generic formula can be attached to the free amine of theN-terminal amino acid by standard methods known in the art. For example,alkyl groups, e.g., (C₁-C₃₀)alkyl, can be attached using reductivealkylation. Hydroxyalkyl groups, e.g., (C₁-C₃₀)hydroxyalkyl, can also beattached using reductive alkylation wherein the free hydroxy group isprotected with a t-butyl ester. Acyl groups, e.g., COE¹, may be attachedby coupling the free acid, e.g., E¹COOH, to the free amine of theN-terminal amino acid by mixing the completed resin with 3 molarequivalents of both the free acid and diisopropylcarbodiimide inmethylene chloride for one hour. If the free acid contains a freehydroxy group, e.g., p-hydroxyphenylpropionic acid, then the couplingshould be performed with an additional 3 molar equivalents of HOBT.

When R¹ is NH—X²—CH₂—CONH₂ (i.e., Z⁰=CONH₂), the synthesis of thepeptide starts with BocHN—X²—CH₂—COOH which is coupled to the MBHAresin. If R¹ is NH—X²—CH₂—COOH (i.e., Z⁰=COOH) the synthesis of thepeptide starts with Boc-HN—X²—CH₂—COOH which is coupled to PAM resin.

The following describes a synthetic method for making a peptide of thisinvention, which method is well-known to those skilled in the art. Othermethods are also known to those skilled in the art.

Benzhydrylamine-polystyrene resin (Advanced ChemTech, Inc., Louisville,Ky.) (0.9 g, 0.3 mmole) in the chloride ion form is placed in a reactionvessel of an Advanced ChemTech Peptide Synthesizer Model 200 programmedto perform the following reaction cycle: (a) methylene chloride; (b) 33%trifluoroacetic acid in methylene chloride (2 times for 1 and 15 mineach); (c) methylene chloride; (d) ethanol; (e) methylene chloride; (f)10% diisopropylethylamine in methylene chloride.

The neutralized resin is stirred with Boc-protected amino acid which isto be the C-terminal amino acid of the desired peptide to be synthesizedand diisopropylcarbodiimide (3 mmole each) in methylene chloride for 1hour and the resulting amino acid resin is then cycled through steps (a)through (f) in the above wash program. The other amino acids (3 mmol) ofthe desired peptide are then coupled successively by the same procedure.The finished peptide is cleaved from the resin by mixing it with anisole(5 ml), dithiothreitol (100 mg) and anhydrous hydrogen fluoride (35 ml)at about 0° C. and stirring it for about 45 min. Excess hydrogenfluoride is evaporated rapidly under a stream of dry nitrogen and freepeptide precipitated and washed with ether. The crude peptide is thendissolved in a minimum volume of dilute acetic acid and eluted on acolumn (2.5×25 cm) of VYDAC® octadecylsilane silica (10 mM) and elutedwith a linear gradient of 20-60% acetonitrile over about 1 h in 0.1%trifluoroacetic acid in water. Fractions are examined by thin layerchromatography and analytical high performance liquid chromatography(40-70% B at 1%/min, solution B is 80% acetonitrile/water containing0.1% TFA) and pooled to give maximum purity rather than yield. Repeatedlyophilization of the solution from water gives the product as a white,fluffy powder.

The product peptide is analyzed by HPLC. Amino acid analysis of an acidhydrolysate of the product peptide can confirm the composition of thepeptide. Laser desorption MS is used to determine the molecular weightof the peptide.

The protected amino acid1-[N-tert-butoxycarbonyl-amino]-1-cyclohexane-carboxylic acid(Boc-A6c-OH) was synthesized as follows. 19.1 g (0.133 mol) of1-amino-1-cyclohexanecarboxylic acid (Acros Organics, Fisher Scientific,Pittsburgh, Pa.) was dissolved in 200 ml of dioxane and 100 ml of water.To it was added 67 ml of 2N NaOH. The solution was cooled in anice-water bath. 32.0 g (0.147 mol) of di-tert-butyl-dicarbonate wasadded to this solution. The reaction mixture was stirred overnight atroom temperature. Dioxane was then removed under reduced pressure. 200ml of ethyl acetate was added to the remaining aqueous solution. Themixture was cooled in an ice-water bath. The pH of the aqueous layer wasadjusted to about 3 by adding 4N HCl. The organic layer was separated.The aqueous layer was extracted with ethyl acetate (1×100 ml). The twoorganic layers were combined and washed with water (2×150 ml), driedover anhydrous MgSO₄, filtered, and concentrated to dryness underreduced pressure. The residue was recrystallized in ethylacetate/hexanes. 9.2 g of the pure product was obtained. 29% yield.

Boc-A5c-OH was synthesized in an analogous manner to that of Boc-A6c-OH.Other protected Acc amino acids can be prepared in an analogous mannerby a person of ordinary skill in the art as enabled by the teachingsherein.

In the synthesis of a peptide of this invention containing A5c, A6cand/or Aib, the coupling time is about 2 hrs. for these residues and theresidue immediately following them. For the synthesis of[Tma-His⁷]hGLP-1(7-36)NH₂ (SEQ ID NO: 117), HBTU (2 mmol) and DIEA (1.0ml) in 4 ml DMF were used to react with the N-terminal free amine of thepeptide-resin in the last coupling reaction; the coupling time is about2 hours.

The full names for the abbreviations used above are as follows: Boc fort-butyloxycarbonyl, HF for hydrogen fluoride, Fm for formyl, Xan forxanthyl, Bzl for benzyl, Tos for tosyl, DNP for 2,4-dinitrophenyl, DMFfor dimethylformamide, DCM for dichloromethane, HBTU for2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate, DIEA for diisopropylethylamine, HOAc for aceticacid, TFA for trifluoroacetic acid, 2CIZ for 2-chlorobenzyloxycarbonyl,2BrZ for 2-bromobenzyloxycarbonyl and OcHex for O-cyclohexyl.

The substituents R² and R³ of the above generic formula can be attachedto the free amine of the N-terminal amino acid by standard methods knownin the art. For example, alkyl groups, e.g., (C₁-C₃₀)alkyl, may beattached using reductive alkylation. Hydroxyalkyl groups, e.g.,(C₁-C₃₀)hydroxyalkyl, can also be attached using reductive alkylationwherein the free hydroxy group is protected with a t-butyl ester. Acylgroups, e.g., COX¹, can be attached by coupling the free acid, e.g.,X¹COOH, to the free amine of the N-terminal amino acid by mixing thecompleted resin with 3 molar equivalents of both the free acid anddiisopropylcarbodiimide in methylene chloride for about one hour. If thefree acid contains a free hydroxy group, e.g., p-hydroxyphenylpropionicacid, then the coupling should be performed with an additional 3 molarequivalents of HOBT.

A compound of the present invention can be tested for activity as aGLP-1 binding compound according to the following procedure.

Cell Culture:

RIN 5F rat insulinoma cells (ATCC-# CRL-2058, American Type CultureCollection, Manassas, Va.), expressing the GLP-1 receptor, were culturedin Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calfserum, and maintained at about 37° C. in a humidified atmosphere of 5%CO₂/95% air.

Radioligand Binding:

Membranes were prepared for radioligand binding studies byhomogenization of the RIN cells in 20 ml of ice-cold 50 mM Tris-HCl witha Brinkman Polytron (Westbury, N.Y.) (setting 6, 15 sec). Thehomogenates were washed twice by centrifugation (39,000 g/10 min), andthe final pellets were re-suspended in 50 mM Tris-HCl, containing 2.5 mMMgCl₂, 0.1 mg/ml bacitracin (Sigma Chemical, St. Louis, Mo.), and 0.1%BSA. For assay, aliquots (0.4 ml) were incubated with 0.05 nM[¹²⁵I]GLP-1(7-36) (SEQ ID NO: 151) (˜2200 Ci/mmol, New England Nuclear,Boston, Mass.), with and without 0.05 ml of unlabeled competing testpeptides. After a 100 min incubation (25° C.), the bound[¹²⁵I]GLP-1(7-36) (SEQ ID NO: 151) was separated from the free by rapidfiltration through GF/C filters (Brandel, Gaithersburg, Md.), which hadbeen previously soaked in 0.5% polyethyleneimine. The filters were thenwashed three times with 5 ml aliquots of ice-cold 50 mM Tris-HCl, andthe bound radioactivity trapped on the filters was counted by gammaspectrometry (Wallac L K B, Gaithersburg, Md.). Specific binding wasdefined as the total [¹²⁵I]GLP-1(7-36) (SEQ ID NO: 151) bound minus thatbound in the presence of 1000 nM GLP-1(7-36) (SEQ ID NO: 3) (Bachem,Torrence, Calif.).

The peptides of this invention can be provided in the form ofpharmaceutically acceptable salts. Examples of such salts include, butare not limited to, those formed with organic acids (e.g., acetic,lactic, maleic, citric, malic, ascorbic, succinic, benzoic,methanesulfonic, toluenesulfonic, or pamoic acid), inorganic acids(e.g., hydrochloric acid, sulfuric acid, or phosphoric acid), andpolymeric acids (e.g., tannic acid, carboxymethyl cellulose, polylactic,polyglycolic, or copolymers of polylactic-glycolic acids). A typicalmethod of making a salt of a peptide of the present invention is wellknown in the art and can be accomplished by standard methods of saltexchange. Accordingly, the TFA salt of a peptide of the presentinvention (the TFA salt results from the purification of the peptide byusing preparative HPLC, eluting with TFA containing buffer solutions)can be converted into another salt, such as an acetate salt bydissolving the peptide in a small amount of 0.25 N acetic acid aqueoussolution. The resulting solution is applied to a semi-prep HPLC column(Zorbax, 300 SB, C-8). The column is eluted with (1) 0.1N ammoniumacetate aqueous solution for 0.5 hrs., (2) 0.25N acetic acid aqueoussolution 0.5 hrs. and (3) a linear gradient (20% to 100% of solution Bover 30 min.) at a flow rate of 4 ml/min (solution A is 0.25N aceticacid aqueous solution; solution B is 0.25N acetic acid inacetonitrile/water, 80:20). The fractions containing the peptide arecollected and lyophilized to dryness.

As is well known to those skilled in the art, the known and potentialuses of GLP-1 is varied and multitudinous [See, Todd, J. F., et al.,Clinical Science, 1998, 95, pp. 325-329; and Todd, J. F. et al.,European Journal of Clinical Investigation, 1997, 27, pp. 533-536].Thus, the administration of the compounds of this invention for purposesof eliciting an agonist effect can have the same effects and uses asGLP-1 itself. These varied uses of GLP-1 may be summarized as follows,treatment of: Type I diabetes, Type II diabetes, obesity, glucagonomas,secretory disorders of the airway, metabolic disorder, arthritis,osteoporosis, central nervous system diseases, restenosis andneurodegenerative diseases. GLP-1 analogues of the present inventionthat elicit an antagonist effect from a subject can be used for treatingthe following: hypoglycemia and malabsorption syndrome associated withgastroectomy or small bowel resection.

Accordingly, the present invention includes within its scopepharmaceutical compositions comprising, as an active ingredient, atleast one of the compounds of formula (I) in association with apharmaceutically acceptable carrier or diluent.

The dosage of active ingredient in the compositions of this inventionmay be varied; however, it is necessary that the amount of the activeingredient be such that a suitable dosage form is obtained. The selecteddosage depends upon the desired therapeutic effect, on the route ofadministration, and on the duration of the treatment. In general, aneffective dosage for the activities of this invention is in the range of1×10⁻⁷ to 200 mg/kg/day, preferably 1×10⁻⁴ to 100 mg/kg/day, which canbe administered as a single dose or divided into multiple doses.

The compounds of this invention can be administered by oral, parenteral(e.g., intramuscular, intraperitoneal, intravenous or subcutaneousinjection, or implant), nasal, vaginal, rectal, sublingual or topicalroutes of administration and can be formulated with pharmaceuticallyacceptable carriers to provide dosage forms appropriate for each routeof administration.

Solid dosage forms for oral administration include capsules, tablets,pills, powders and granules. In such solid dosage forms, the activecompound is admixed with at least one inert pharmaceutically acceptablecarrier such as sucrose, lactose, or starch. Such dosage forms can alsocomprise, as is normal practice, additional substances other than suchinert diluents, e.g., lubricating agents such as magnesium stearate. Inthe case of capsules, tablets and pills, the dosage forms may alsocomprise buffering agents. Tablets and pills can additionally beprepared with enteric coatings.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, the elixirscontaining inert diluents commonly used in the art, such as water.Besides such inert diluents, compositions can also include adjuvants,such as wetting agents, emulsifying and suspending agents, andsweetening, flavoring and perfuming agents.

Preparations according to this invention for parenteral administrationinclude sterile aqueous or non-aqueous solutions, suspensions, oremulsions. Examples of non-aqueous solvents or vehicles are propyleneglycol, polyethylene glycol, vegetable oils, such as olive oil and cornoil, gelatin, and injectable organic esters such as ethyl oleate. Suchdosage forms may also contain adjuvants such as preserving, wetting,emulsifying, and dispersing agents. They may be sterilized by, forexample, filtration through a bacteria-retaining filter, byincorporating sterilizing agents into the compositions, by irradiatingthe compositions, or by heating the compositions. They can also bemanufactured in the form of sterile solid compositions which can bedissolved in sterile water, or some other sterile injectable mediumimmediately before use.

Compositions for rectal or vaginal administration are preferablysuppositories which may contain, in addition to the active substance,excipients such as coca butter or a suppository wax.

Compositions for nasal or sublingual administration are also preparedwith standard excipients well known in the art.

Further, a compound of this invention can be administered in a sustainedrelease composition such as those described in the following patents andpatent applications. U.S. Pat. No. 5,672,659 teaches sustained releasecompositions comprising a bioactive agent and a polyester. U.S. Pat. No.5,595,760 teaches sustained release compositions comprising a bioactiveagent in a gelable form. U.S. application Ser. No. 08/929,363 filed Sep.9, 1997, teaches polymeric sustained release compositions comprising abioactive agent and chitosan. U.S. application Ser. No. 08/740,778 filedNov. 1, 1996, teaches sustained release compositions comprising abioactive agent and cyclodextrin. U.S. application Ser. No. 09/015,394filed Jan. 29, 1998, teaches absorbable sustained release compositionsof a bioactive agent. U.S. application Ser. No. 09/121,653 filed Jul.23, 1998, teaches a process for making microparticles comprising atherapeutic agent such as a peptide in an oil-in-water process. U.S.application Ser. No. 09/131,472 filed Aug. 10, 1998, teaches complexescomprising a therapeutic agent such as a peptide and a phosphorylatedpolymer. U.S. application Ser. No. 09/184,413 filed Nov. 2, 1998,teaches complexes comprising a therapeutic agent such as a peptide and apolymer bearing a non-polymerizable lactone. The teachings of theforegoing patents and applications are incorporated herein by reference.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Also, all publications, patentapplications, patents and other references mentioned herein areincorporated by reference.

The following examples describe synthetic methods for making a peptideof this invention, which methods are well-known to those skilled in theart. Other methods are also known to those skilled in the art. Theexamples are provided for the purpose of illustration and is not meantto limit the scope of the present invention in any manner.

EXAMPLE 1 [D-Ala⁸, Ala^(17,22,23,27), 3-Pal^(19,31),Gaba³⁴]-GLP-1(7-34)NH₂

Benzhydrylamine-polystyrene resin (Advanced ChemTech, Inc. Louisville,Ky.) (0.9 g, 0.3 mmole) in the chloride ion form was placed in areaction vessel of an Advanced ChemTech peptide synthesizer Model 200programmed to perform the following reaction cycle: (a) methylenechloride; (b) 33% trifluoroacetic acid in methylene chloride (2 timesfor 1 and 15 min each); (c) methylene chloride; (d) ethanol; (e)methylene chloride; (f) 10% diisopropylethylamine in methylene chloride.

The neutralized resin was stirred with Boc-Gaba anddiisopropylcarbodiimide (3 mmole each) in methylene chloride for 1 hourand the resulting amino acid resin was then cycled through steps (a) to(f) in the above wash program. The following amino acids (3 mmole) werethen coupled successively by the same procedure: Boc-Val, Boc-Leu,Boc-3-Pal, Boc-Ala, Boc-Ile, Boc-Phe, Boc-Ala, Boc-Lys(2-Cl-Z), Boc-Ala,Boc-Ala, Boc-Ala, Boc-Ala, Boc-Glu(Bzl), Boc-Leu, Boc-3-Pal,Boc-Ser(Bzl), Boc-Ala, Boc-Val, Boc-Asp(Bzl), Boc-Ser(Bzl),Boc-Thr(Bzl), Boc-Phe, Boc-Thr(Bzl), Boc-Gly, Boc-Glu(Bzl), Boc-D-Ala,Boc-His(Bom).

The resin with the completed peptide sequence was mixed with anisole (5ml), dithiothreitol (100 mg) and anhydrous hydrogen fluoride (35 ml) atabout 0° C. and stirred for about 45 min. Excess hydrogen fluoride wasevaporated rapidly under a stream of dry nitrogen and free peptideprecipitated and washed with ether. The crude peptide was then dissolvedin a minimum volume of dilute acetic acid and eluted on a column (2.5×25cm) of VYDAC® octadecylsilane silica (10 mM) and eluted with a lineargradient of 20-60% acetonitrile over about 1 h in 0.1% trifluoroaceticacid in water. Fractions were examined by thin layer chromatography andanalytical high performance liquid chromatography (40-70% B at 1%/min;r.t.: 14.1 min) and pooled to give maximum purity rather than yield.Repeated lyophilization of the solution from water gives the product(49.9 mg) as a white, fluffy powder.

The product was found to be homogeneous by HPLC and tlc. Amino acidanalysis of an acid hydrolysate confirms the composition of the peptide.Laser desorption MS gave a MW of 2880 (Calc. M+H 2873).

EXAMPLE 2 Synthesis of Peptide Lower-Alkylamides

Peptides are assembled on O-benzyl-polystyrene resin (often referred toas Merrifield resin) using the Boc amino acid protocol described inExample 1, except that Asp and Glu amino acid carboxyl side-chains areprotected with an Fm (fluorenylmethyl ester) group. Completedpeptide-resins are suspended in dilute DMF solutions of an appropriatelower alkylamine (such as ethylamine, propylamine, phenethylamine,1,2-diaminoethane, etc.) and stirred at about 60° C. (for about 18 hrs)whereupon filtration, removal of solvents under reduced pressure andtrituration of cleaved peptide oil with ether gives a solid, protectedalkylamide peptide. This is then subjected to HF cleavage to removeadditional side chain protecting groups and HPLC purification asdescribed in Example 1.

EXAMPLES 3-5

Examples 3-5 can be synthesized substantially according to the proceduredescribed in Example 1 using the appropriate protected amino acids toyield the noted peptides.

Example 3: [Aib⁸, D-Ala¹⁷, Ala^(18,22,23,27), 3-Pal^(19,31), Tle¹⁶,Gaba³⁴]-GLP-1(7-34)NH₂ Example 4: [Aib³, D-Ala¹⁷, Ala^(22,23,27),3-Pal^(19,31), Tle¹⁶]-GLP-1(7-33)NH₂ Example 5: [Aib⁸ D-Ala¹⁷Ala^(22,23,27), 3-Pal^(19,31), Tle^(16,20)]-GLP-1(7-33)NH₂ EXAMPLES 6-51

Examples 6-51 were made substantially according to the proceduredescribed for Example 1 but using the appropriate protected amino acidto yield the noted peptide. MS were obtained by laser desorption MS (NAmeans not available).

Example 6: [D-Ala^(8,23,27), 3-Pal^(19,31)]hGLP-1(7-35)-NH₂; MS=2971.0;Calc. MW=2974.4.Example 7: [Ala^(18,23,27), 3-Pal^(19,31)]hGLP-1(7-35)-NH₂ (SEQ ID NO:83); MS=2954.4; Calc. MW=2958.4.Example 8: [Ala^(16,23,27), 3-Pal^(19,31)]hGLP-1(7-35)-NH₂ (SEQ ID NO:84); MS=2943.0; Calc. MW=2946.3.Example 9: [Ala^(14,23,27), 3-Pal^(19,31)]hGLP-1(7-35)-NH₂ (SEQ ID NO:85); MS=2956.0; Calc. MW=2958.4.Example 10: [Ala^(22,23,27), 3-Pal^(19,31)]hGLP-1(7-35)-NH₂ (SEQ ID NO:86); MS=2981.0; Calc. MW=2988.4.Example 11: [Hppa⁷]hGLP-1(7-36)-NH₂ (SEQ ID NO: 87); MS=NAExample 12: [Ala^(15,23,27), 3-Pal^(19,31)]hGLP-1(7-35)-NH₂ (SEQ ID NO:88); MS=2928.0; Calc. MW=2930.4.Example 13: [Ala^(17,23,27), 3-Pal^(19,31)]hGLP-1(7-35)-NH₂ (SEQ ID NO:89); MS=2955.0; Calc. MW=2958.4.Example 14: [Ala^(22,23,27), 3-Pal^(19,31), Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQID NO: 90); MS=2896.0; Calc. MW=2888.3.Example 15: [Ala^(15,22,23,27), 3-Pal^(19,31), Gaba³⁴]hGLP-1(7-34)-NH₂(SEQ ID NO: 91); MS=2852.0; Calc. MW=2844.3.Example 16: [Ala^(17,22,23,27), 3-Pal^(19,31), Gaba³⁴]hGLP-1(7-34)-NH₂(SEQ ID NO: 92); MS=2880.0; Calc. MW=2872.3.Example 17: [Ala^(18,22,23,27), 3-Pal^(19,31), Gaba³⁴]hGLP-1(7-34)-NH₂(SEQ ID NO: 93); MS=2870.0; Calc. MW=2872.3.Example 18: [Ala^(21,22,23,27), 3-Pal^(19,31), Gaba³⁴]hGLP-1(7-34)-NH₂(SEQ ID NO: 94); MS=NA.Example 19: [Ala^(22,23,26,27), 3-Pal^(19,31), Gaba³⁴]hGLP-1(7-34)-NH₂(SEQ ID NO: 95); MS=2832.0; Calc. MW=2831.2.Example 20: [Ala^(22,23,27,32), 3-Pal^(19,31), Gaba³⁴]hGLP-1(7-34)-NH₂(SEQ ID NO: 96); MS=2855.0; Calc. MW=2846.2.Example 21: [Ala^(22,23,26,27), 3-Pal^(19,31), Gaba³³]hGLP-1(7-33)-NH₂(SEQ ID NO: 97); MS=2729.0; Calc. MW=2732.0.Example 22: [Ala^(22,23,27,31), 3-Pal¹⁹, Gaba³³]hGLP-1(7-33)-NH₂ (SEQ IDNO: 98); MS=2711.6; Calc. MW=2712.0.Example 23: [Ala^(22,23,27,28), 3-Pal^(19,31), Gaba³³]hGLP-1(7-33)-NH₂(SEQ ID NO: 99); MS=2712.0; Calc. MW=2713.0.Example 24: [Ala^(22,23,27,29), 3-Pal^(19,31), Gaba³³]hGLP-1(7-33)-NH₂(SEQ ID NO: 100); MS=2746.9; Calc. MW=2747.1.Example 25: [Ala^(23,27), 3-Pal^(19,31), Gaba³³]hGLP-1(7-33)-NH₂ (SEQ IDNO: 101); MS=2777.0; Calc. MW=2,775.1.Example 26: [Ala^(20,22,23,27), 3-Pal^(19,31), Gaba³³]hGLP-1(7-33)-NH₂(SEQ ID NO: 102); MS=2742.0; Calc. MW=2747.1.Example 27: [Ala^(22,23,27), 3-Pal^(19,31), Gaba³³]hGLP-1(7-33)-NH₂ (SEQID NO: 103); MS=2786.7; Calc. MW=2789.1.Example 28: [Ala^(17,22,23,27), 3-Pal^(19,31), Gaba³³]hGLP-1(7-33)-NH₂(SEQ ID NO: 104); MS=2771.0; Calc. MW=2773.1.Example 29: [D-Ala¹⁰, Ala^(22,23,27), 3-Pal^(19,31),Gaba³³]hGLP-1(7-33)-NH₂; MS=2802.0; Calc. MW=2803.2.Example 30: [D-Ala⁸, Ala^(17,23,27), 3-Pal^(19,31)]hGLP-1(7-34)-NH₂;MS=2905.0; Calc. MW=2901.3.Example 31: [Ala^(17,23,27), 3-Pal^(19,26,31)]hGLP-1(7-34)-NH₂ (SEQ IDNO: 105); MS=2920.0; Calc. MW=2921.3.Example 32: [D-Ala⁸, Ala¹⁷, 3-Pal^(19,31)]hGLP-1(7-34)-NH₂; MS=2908.0(Na⁺ salt); Calc. MW=2885.3.Example 33: [Ala^(17,23,27), 3-Pal^(19,31)]hGLP-1(7-34)-NH₂ (SEQ ID NO:106); MS=2907.0; Calc. MW=2901.3.Example 34: [D-Ala⁸, Ala^(17,23,27), 3-Pal^(19,31),Tle²⁹]hGLP-1(7-34)-NH₂; MS=2906.0; Calc. MW=2901.3.Example 35: [D-Ala⁸, Ala^(17,23,27), 3-Pal^(19,31),Tle¹⁶]hGLP-1(7-34)-NH₂; MS=2914.0; Calc. MW=2915.4.Example 36: [D-Ala⁸, Ala^(17,23,27), 3-Pal^(19,31),Gaba³⁴]hGLP-1(7-34)-NH₂; MS=2856.8; Calc. MW=2858.2.Example 37: [D-Ala²², Ala^(17,23,27), 3-Pal^(19,31),Gaba³⁴]hGLP-1(7-34)-NH₂; MS=2871.0; Calc. MW=2872.3.Example 38: [Aib⁸, Ala^(17,23,27), 3-Pal^(19,31),Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 107); MS=2875.0; Calc. MW=2872.3.Example 39: [D-Ala⁸, Ala^(17,22,23,27), 3-Pal^(19,31)]hGLP-1(7-33)-NH₂;MS=2786.0; Calc. MW=2787.2.Example 40: [Aib⁸ Ala^(17,22,23,27), 3-Pal^(19,31)]hGLP-1(7-33)-NH₂ (SEQID NO: 108); MS=2800.0; Calc. MW=2801.2.Example 41: [Ala^(17,18,23,27), 3-Pal^(19,31), Gaba³⁴]hGLP-1(7-34)-NH₂(SEQ ID NO: 109); MS=2842.5; Calc. MW=2842.2.Example 42: [Ala^(17,23,27), 3-Pal^(19,31), Tle³³,Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 110); MS=2871.0; Calc. MW=2872.3.Example 43: [Tle¹⁶, Ala^(17,23,27), 3-Pal^(19,31),Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 111); MS=2870.0; Calc. MW=2872.3.Example 44: [N-Me-D-Ala⁸, Ala^(17,22,23,27),3-Pal^(19,31)]hGLP-1(7-33)-NH₂; MS=2795.0; Calc. MW=2801.2.Example 45: [Aib⁸, Ala^(17,18,22,23,27), 3-Pal^(19,31)]hGLP-1(7-33)-NH₂(SEQ ID NO: 112); MS=2784.2; Calc. MW=2785.2.Example 46: [Ala^(17,18,22,23,27), 3-Pal^(19,31), Tle^(16,20)Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 113); MS=2871.9; Calc. MW=2870.3.Example 47: [D-Ala⁸, Ala^(17,18,22,23,27), 3-Pal^(19,31), Tle¹⁶,Gaba³⁴]hGLP-1(7-34)-NH₂; MS=2870.0; Calc. MW=2870.3.Example 48: [D-Ala^(8,22), Ala^(17,18,23,27), 3-Pal^(19,31),Gaba³⁴]hGLP-1(7-34)-NH₂; MS=2856.3; Calc. MW=2856.3.Example 49: [D-Ala^(8,18), Ala^(17,22,23,27), 3-Pal^(19,31),Gaba³⁴]hGLP-1(7-34)-NH₂; MS=NA.Example 50: [D-Ala⁸, Ala^(17,18,22,23,27), 3-Pal^(19,31),Gaba³⁴]hGLP-1(7-34)-NH₂; MS=NA.Example 51: [D-Ala⁸, Ala^(17,18,22,23,27), 3-Pal^(19,31),Gaba³⁴]hGLP-1(7-34)-NH₂; MS=2861.6; Calc. MW=2856.3.

EXAMPLE 52 [Aib⁸, A6c³²]hGLP-1(7-36)NH₂ (SEQ ID NO: 114)

The title peptide was synthesized on an Applied Biosystems (Foster City,Calif.) model 430A peptide synthesizer which was modified to doaccelerated Boc-chemistry solid phase peptide synthesis. See Schnolzer,et al., Int. J. Peptide Protein Res., 40:180 (1992).4-Methylbenzhydrylamine (MBHA) resin (Peninsula, Belmont, Calif.) withthe substitution of 0.91 mmol/g was used. The Boc amino acids (Bachem,Calif., Torrance, Calif.; Nova Biochem., LaJolla, Calif.) were used withthe following side chain protection: Boc-Ala-OH, Boc-Arg(Tos)-OH,Boc-Asp(OcHex)-OH, Boc-Tyr(2BrZ)-OH, Boc-His(DNP)—OH, Boc-Val-OH,Boc-Leu-OH, Boc-Gly-OH, Boc-Gln-OH, Boc-Ile-OH, Boc-Lys(2CIZ)-OH,Boc-Thr(Bzl)-OH, Boc-A6c-OH, Ser(Bzl)-OH, Boc-Phe-OH, Boc-Aib-OH,Boc-Glu(OcHex)-OH and Boc-Trp(Fm)—OH. The synthesis was carried out on a0.20 mmol scale. The Boc groups were removed by treatment with 100% TFAfor 2×1 min. Boc amino acids (2.5 mmol) were pre-activated with HBTU(2.0 mmol) and DIEA (1.0 ml) in 4 ml of DMF and were coupled withoutprior neutralization of the peptide-resin TFA salt. Coupling times wereabout 5 min except for the Boc-Aib-OH and Boc-A6c-OH residues and thefollowing residues, Boc-Trp(Fm)-OH and Boc-His(DNP)—OH wherein thecoupling times were about 2 hours.

At the end of the assembly of the peptide chain, the resin was treatedwith a solution of 20% mercaptoethanol/10% DIEA in DMF for 2×30 min toremove the DNP group on the His side chain. The N-terminal Boc group wasthen removed by treatment with 100% TFA for 2×2 min. Afterneutralization of the peptide-resin with 10% DIEA in DMF (1×1 min), theformyl group on the side of the chain of Trp was removed by treatmentwith a solution of 15% ethanolamine/15% water/70% DMF for 2×30 min. Thepartially-deprotected peptide-resin was washed with DMF and DCM anddried under reduced pressure. The final cleavage was done by stirringthe peptide-resin in 10 ml of HF containing 1 ml of anisole anddithiothreitol (24 mg) at 0° C. for about 75 min. HF was removed with aflow of nitrogen. The residue was washed with ether (6×10 ml) andextracted with 4N HOAc (6×10 ml).

The peptide mixture in the aqueous extract was purified on areverse-phase preparative high pressure liquid chromatography (HPLC)using a reverse phase VYDAC® C₁₈ column (Nest Group, Southborough,Mass.). The column was eluted with a linear gradient (20% to 50% ofsolution B over 105 min) at a flow rate of 10 ml/min (Solution A=watercontaining 0.1% TFA; Solution B=acetonitrile containing 0.1% of TFA).Fractions were collected and checked on analytical HPLC. Thosecontaining pure product were combined and lyophilized to dryness. 92 mgof a white solid was obtained. Purity was >99% based on analytical HPLCanalysis. Electro-spray mass spectrometer analysis gave the molecularweight at 3324.2 (the calculated molecular weight is 3323.7).

The synthesis of other compounds of the present invention can be carriedout in the same manner as described for the synthesis of [Aib⁸,A6c³²]hGLP-1(7-36)NH₂ (SEQ ID NO: 114) in Example 52 above but using theappropriate protected amino acids depending on the desired peptide.

[(N^(α)-HEPES-His)₇]hGLP-1(7-36)NH₂ (SEQ ID NO: 152) {HEPES is(4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid)} can besynthesized as follows: After assembly of the peptide long chain on MBHAresin (0.20 mmol), the peptide-resin is treated with 100% TFA (2×2 min.)and washed with DMF and DCM. The resin is then neutralized with 10% DIEAin DMF for about 2 min. After washing with DMF and DCM, the resin istreated with 0.23 mmol of 2-chloro-1-ethanesulfonyl chloride and 0.7mmol of DIEA in DMF for about 1 hour. The resin is washed with DMF andDCM and treated with 1.2 mmol of 2-hydroxyethylpiperazine for about 2hours. The resin is washed with DMF and DCM and treated with differentreagents ((1) 20% mercaptoethanol/10% DIEA in DMF and (2) 15%ethanolamine/15% water/70% DMF) to remove the DNP group from the Hisside chain and formyl group on the Trp side chain as described abovebefore the final HF cleavage of the peptide from the resin.

[(N^(α)-HEPA-His)₇]hGLP-1(7-36)NH₂ (SEQ ID NO: 153)([(4-(2-hydroxyethyl)-1-piperazineacetyl)-His⁷]hGLP-1(7-36)NH₂) can bemade substantially according to the procedure described immediatelyabove for making [(N^(α)-HEPES-His)⁷]hGLP-1(7-36)NH₂ (SEQ ID NO: 152)except that 2-bromo-acetic anhydride is used in place of2-chloro-1-ethanesulfonyl chloride.

EXAMPLES 53-90 and 104

Examples 53-90 and 104 were made substantially according to Example 52but using the appropriate protected amino acid.

Example 53: [A6c^(20,32)]hGLP-1(7-36)-NH₂ (SEQ ID NO:115); MS=3322.3;Calc. MW=3321.7.Example 54: [Aib⁸]hGLP-1(7-36)-NH₂ (SEQ ID NO: 116); MS=3311.7; Calc.MW=3311.7.Example 55: [(Tma-His)⁷]hGLP-1(7-36)-NH₂ (SEQ ID NO: 117); MS=3395.9;Calc. MW=3396.9.Example 56: [A6c⁸]hGLP-1(8-36)-NH₂ (SEQ ID NO: 118); MS=3214.5; Calc.MW=3214.7.Example 57: [A6c⁸]hGLP-1(7-36)-NH₂ (SEQ ID NO:119); MS=3351.5; Calc.MW=3351.8.Example 58: [A6c^(16,20)]hGLP-1(7-36)-NH₂ (SEQ ID NO: 120); MS=3335.9;Calc. MW=3335.8.Example 59: [A6c^(29,32)]hGLP-1(7-36)-NH₂ (SEQ ID NO: 121); MS=3321.7;Calc. MW=3321.7.Example 60: [A6c²⁰, Aib²⁴]hGLP-1(7-36)-NH₂ (SEQ ID NO: 122); MS=3323.6;Calc. MW=3323.7.Example 61: [Aib²⁴, A6c^(29,32)]hGLP-1(7-36)-NH₂ (SEQ ID NO: 123);MS=3335.7; Calc. MW=3335.8.Example 62: [A6c^(16,29,32)]hGLP-1(7-36)-NH₂ (SEQ ID NO: 124);MS=3347.7; Calc. MW=3347.8.Example 63: [Ura⁷]hGLP-1(7-36)-NH₂ (SEQ ID NO: 125); MS=3279.5; Calc.MW=3280.7.Example 64: [Paa⁷]hGLP-1(7-36)-NH₂ (SEQ ID NO: 126); MS=3290.9; Calc.MW=3291.8.Example 65: [Pta⁷]hGLP-1(7-36)-NH₂ (SEQ ID NO: 127); MS=3311.2; Calc.MW=3311.8.Example 66: [N-Me-Ala⁸]hGLP-1(7-36)-NH₂ (SEQ ID NO: 128); MS=3311.4;Calc. MW=3311.7.Example 67: [N-Me-D-Ala⁸]hGLP-1(7-36)-NH₂; MS=3311.6; Calc. MW=3311.7.Example 68: [N-Me-D-Ala⁸]hGLP-1(8-36)-NH₂; MS=3174.0; Calc. MW=3174.6.Example 69: [N-Me-Gly⁸]hGLP-1(7-36)-NH₂ (SEQ ID NO: 129); MS=3297.3;Calc. MW=3297.7.Example 70: [A5c⁸]hGLP-1(7-36)-NH₂ (SEQ ID NO: 130); MS=3337.3; Calc.MW=3337.8.Example 71: [N-Me-Glu⁹]hGLP-1(7-36)-NH₂ (SEQ ID NO: 131); MS=3311.4;Calc. MW=3311.7.Example 72; [A5c⁸, A6c^(20,32)]hGLP-1(7-36)-NH₂ (SEQ ID NO: 132);MS=3361.4; Calc. MW=3361.8.Example 73: [Aib⁸, A6c³²]hGLP-1(7-36)-NH₂ (SEQ ID NO: 133); MS=3323.2;Calc. MW=3323.7.Example 74: [Aib^(8,25)]hGLP-1(7-36)-NH₂ (SEQ ID NO: 134); MS=3325.8;Calc. MW=3325.7.Example 75: [Aib^(8,24)]hGLP-1(7-36)-NH₂ (SEQ ID NO: 135); MS=3325.8;Calc. MW=3325.7.Example 76: [Aib^(8,30)]hGLP-1(7-36)-NH₂ (SEQ ID NO: 136); MS=3326.1;Calc. MW=3325.7.Example 77: [Aib⁸, Cha²⁰]hGLP-1(7-36)-NH₂ (SEQ ID NO: 137); MS=3351.8;Calc. MW=3351.8.Example 78: [Aib⁸, Cha³²]hGLP-1(7-36)-NH₂ (SEQ ID NO: 138); MS=3352.0;Calc. MW=3351.8.Example 79: [Aib⁸, Glu²³]hGLP-1(7-36)-NH₂ (SEQ ID NO: 139); MS=3311.7;Calc. MW=3312.7.Example 80: [Aib⁸, A6c²⁰]hGLP-1(7-36)-NH₂ (SEQ ID NO: 140); MS=3323.6;Calc. MW=3323.7.Example 81: [Aib⁸, A6c^(20,32)]hGLP-1(7-36)-NH₂ (SEQ ID NO: 141);MS=3335.3; Calc. MW=3335.7.Example 82: [Aib⁸, Lys²⁵]hGLP-1(7-36)-NH₂ (SEQ ID NO: 142); MS=3339.8;Calc. MW=3339.8.Example 83: [Aib⁸, β-Ala²²]hGLP-1(7-36)-NH₂ (SEQ ID NO: 143); MS=3325.6;Calc. MW=3325.8.Example 84: [Aib⁸, Lys²⁵]hGLP-1(7-36)-NH₂ (SEQ ID NO: 144); MS=3369.0;Calc. MW=3368.8.Example 85: [Aib⁸, A6c¹²]hGLP-1(7-36)-NH₂ (SEQ ID NO: 145); MS=3289.8;Calc. MW=3289.7.Example 86: [Aib⁸, A6c²⁹]hGLP-1(7-36)-NH₂ (SEQ ID NO:146); MS=3323.9;Calc. MW=3323.7.Example 87: [Aib⁸, A6c³]hGLP-1(7-36)-NH₂ (SEQ ID NO: 147); MS=3338.0;Calc. MW=3337.8.Example 88: [Aib^(8,14)]hGLP-1(7-36)NH₂ (SEQ ID NO: 148); MS=3309.8;Calc. MW=3309.7.Example 89: [Aib^(8,18)]hGLP-1(7-36)NH₂ (SEQ ID NO: 149); MS=3309.7;Calc. MW=3309.7.Example 90: [Aib^(8,7)]hGLP-1(7-36)NH₂ (SEQ ID NO; 150); MS=3309.4;Calc. MW=3309.7.Example 104: [Aib⁸, D-Arg;²⁶]hGLP-1(7-36)NH₂; MS=3310.7; Calc.MW=3311.73.

EXAMPLE 91 [Aib⁸, A5c³³]hGLP-1(7-36)NH₂ (SEQ ID NO: 154)

The title compound can be made substantially according to Example 52using the appropriate protected amino acids.

EXAMPLE 92 [Aib⁸, A6c³², Lys³⁶(N^(ε)-tetradecanoyl)]hGLP-1(7-36)NH₂ (SEQID NO: 155)

The Boc amino acids to be used are the same as those in the synthesis of[Aib⁸, A6c³²]hGLP-1(7-36)NH₂ (SEQ ID NO: 114) (Example 52) except thatFmoc-Lys(Boc)-OH is used here for the Lys³⁶(N^(ε)-tetradecanoyl)residue. The first amino acid residue is coupled to the resin manuallyon a shaker. 2.5 mmol of Fmoc-Lys(Boc)-OH is dissolved in 4 ml of 0.5NHBTU in DMF. To the solution is added 1 ml of DIEA. The mixture isshaken for about 2 min. To the solution is then added 0.2 mmol of MBHAresin (substitution=0.91 mmol/g). The mixture is shaken for about 1 hr.The resin is washed with DMF and treated with 100% TFA for 2×2 min toremove the Boc protecting group. The resin is washed with DMF. Myristicacid (2.5 mmol) is pre-activated with HBTU (2.0 mmol) and DIEA (1.0 ml)in 4 ml of DMF for 2 min and is coupled to the Fmoc-Lys-resin. Thecoupling time is about 1 hr. The resin is washed with DMF and treatedwith 25% piperidine in DMF for 2×20 min to remove the Fmoc protectinggroup. The resin is washed with DMF and transferred to the reactionvessel of the peptide synthesizer. The remainder of the synthesis andpurification procedures of the peptide are the same as those in thesynthesis of [Aib⁸, A6c³²]hGLP-1(7-36)NH₂. (SEQ ID NO: 114)

The syntheses of other compounds containing Lys(N^(ε)-alkanoyl) residueare carried out in an analogous manner as described for the synthesis of[Aib⁸, A6c³², Lys³⁶(N^(ε)-tetradecanoyl)]hGLP-1(7-36)NH₂ SEQ ID NO:155). Fmoc-Lys(Boc)-OH amino acid is used for the residue ofLys(N^(ε)-alkanoyl) in the peptide, while Boc-Lys(2CIZ)-OH amino acid isused for the residue of Lys. If the Lys(N^(ε)-alkanoyl) residue is notat the C-terminus, the peptide fragment immediately prior to theLys(N^(ε)-alkanoyl) residue is assembled on the resin on the peptidesynthesizer first.

EXAMPLES 93-98

Examples 93-98 can be made substantially according to the proceduredescribed for Example 92 using the appropriate amino acids.

Example 93: [Aib⁸, A6c³², Lys³⁶(N^(ε)-tetradecanoyl)]hGLP-1(7-36)NH₂(SEQ ID NO: 155)Example 94: [Aib⁸, Arg^(26,34), A6c³², Lys³⁶(N^(ε)-tetradecanoyl)]hGLP-1(7-36)NH₂ (SEQ ID NO: 156)Example 95: [Aib⁸, Arg²⁸, A6c³²,Lys³⁴(N^(ε)-tetradecanoyl)]hGLP-1(7-36)NH₂ (SEQ ID NO: 157)Example 96: [Aib⁸, Lys²⁶(N^(ε)-tetradecanoyl), A6c³²,Arg³⁴]hGLP-1(7-36)NH₂ (SEQ ID NO: 158)Example 97: [Aib⁸, Lys³⁶(N^(ε)-octanoyl)]hGLP-1(7-36)NH₂ (SEQ ID NO:159)Example 98: [Aib⁸, A6c^(20,32), Lys³⁶(N^(ε)-octanoyl)]hGLP-1(7-36)NH₂(SEQ ID NO: 160)

EXAMPLE 99 [Aib⁸, Arg^(26,34), A6c³²,Lys³⁶(N^(ε)-tetradecanoyl)]hGLP-1(7-36)-OH (SEQ ID NO: 161)

The Boc amino acids to be used are the same as those used in thesynthesis of [Aib⁸, A6c³², Lys³⁶(N^(ε)-tetradecanoyl)]hGLP-1(7-36)NH₂(SEQ ID NO: 162) (Example 92). Fmoc-Lys(Boc)-OH (2.5 mmol) ispre-activated with HBTU (2.0 mmol), HOBt (2.0 mmol) and DIEA (2.5 ml) inDMF (4 ml) for about 2 min. This amino acid is coupled to 235 mg of PAMresin (Chem-Impex, Wood Dale, Ill.; substitution=0.85 mmol/g) manuallyon a shaker. The coupling time is about 8 hrs. The remainder of thesynthesis and purification procedures for making the peptide are thesame as those described in Example 52.

The syntheses of other analogs of hGLP-1(7-36)-OH (SEQ ID NO: 3) andhGLP-1(7-37)-OH, (SEQ ID NO: 4) which contain Lys(N^(ε)-alkanoyl)residue, are carried out in an analogous manner as described for thesynthesis of [Aib⁸, Arg^(26,34), A6c³²,Lys³⁶(N^(ε)-tetradecanoyl)]hGLP-1(7-36)-OH (SEQ ID NO: 161).Fmoc-Lys(Boc)-OH amino acid is used for the residue ofLys(N^(ε)-alkanoyl) in the peptide, while Boc-Lys(2CIZ)-OH amino acid isused for the residue of Lys.

EXAMPLES 100-103

Examples 100-103 can be made substantially according to the proceduredescribed for Example 99 using the appropriate amino acids.

Example 100: [Aib⁸, Arg²⁶, A6c³²,Lys³⁴(NF-tetradecanoyl)]hGLP-1(7-36)-OH (SEQ ID NO: 162)Example 101: [Aib⁸, Lys²⁶(N^(ε)-tetradecanoyl), A6c³²,Arg³⁴]hGLP-1(7-36)-OH (SEQ ID NO: 163)Example 102: [Aib⁸, Arg^(26,34)-A6c³²,Lys³⁶(N^(ε)-tetradecanoyl)]hGLP-1(7-37)-OH (SEQ ID NO: 164)Example 103: [Aib⁸, Arg²⁶, A6c³²,Lys³⁴(N^(ε)-tetradecanoyl)]hGLP-1(7-37)-OH (SEQ ID NO: 165)

1. A compound of formula (I),(R²R³)-A⁷-A⁸-A⁹-A¹⁰-A¹¹-A¹²-A¹³-A¹⁴-A¹⁵-A¹⁶-A¹⁷-A¹⁸-A¹⁹-A²⁰-A²¹-A²²-A²³-A²⁴-A²⁵-A²⁶-A²⁷-A²⁸-A²⁹-A³⁰-A³¹-A³²-A³³-A³⁴-A³⁵-A³⁶-A³⁷-R¹,  (I)wherein A⁷ is L-His, Ura, Paa, Pta, D-His, Tyr, 3-Pal, 4-Pal, Hppa,Tma-His, Amp or deleted, provided that when A⁷ is Ura, Paa, Pta or Hppathen R² and R³ are deleted; A⁸ is Ala, D-Ala, Aib, Acc, N-Me-Ala,N-Me-D-Ala, Arg or N-Me-Gly; A⁹ is Glu, N-Me-Glu, N-Me-Asp or Asp; A¹⁰is Gly, Acc, Ala, D-Ala, Phe or Aib; A¹¹ is Thr or Ser; A¹² is Phe, Acc,Aic, Aib, 3-Pal, 4-Pal, β-Nal, Cha, Trp or X¹-Phe; A¹³ is Thr or Ser;A¹⁴ is Ser, Thr, Ala or Aib; A¹⁵ is Asp, Ala, D-Asp or Glu; A¹⁶ is Val,D-Val, Acc, Aib, Leu, Ile, Tle, Nle, Abu, Ala, D-Ala, Tba or Cha; A¹⁷ isSer, Ala, D-Ala, Aib, Acc or Thr; A¹³ is Ser, Ala, D-Ala, Aib, Acc orThr; A¹⁹ is Tyr, D-Tyr, Cha, Phe, 3-Pal, 4-Pal, Acc, β-Nal, Amp orX¹-Phe; A²⁰ is Leu, Ala, Acc, Aib, Nle, Ile, Cha, Tle, Val, Phe orX¹-Phe; A²¹ is Glu, Ala or Asp; A²² is Gly, Acc, Ala, D-Ala, β-Ala orAib; A²³ is Gln, Asp, Ala, D-Ala, Aib, Acc, Asn or Glu; A²⁴ is Ala, Aib,Val, Abu, Tle or Acc; A²⁵ is Ala, Aib, Val, Abu, Tle, Acc, Lys, Arg,hArg, Orn, HN—CH((CH₂)_(n)—NR¹⁰R¹¹)—C(O) or HN—CH((CH₂)_(e)—X³)—C(O);A²⁶ is Lys, Ala, 3-Pal, 4-Pal, Arg, hArg, Orn, Amp,HN—CH((CH₂)_(n)—NR¹⁰R¹¹)—C(O) or HN—CH((CH₂)_(e)—X³)—C(O); A²⁷ is Glu,Ala, D-Ala or Asp; A²⁸ is Phe, Ala, Pal, β-Nal, X¹-Phe, Aic, Acc, Aib,Cha or Trp; A²⁹ is Ile, Acc, Aib, Leu, Nle, Cha, Tle, Val, Abu, Ala, Tbaor Phe; A³⁰ is Ala, Aib, Acc or deleted; A³¹ is Trp, Ala, β-Nal, 3-Pal,4-Pal, Phe, Acc, Aib, Cha, Amp or deleted; A³² is Leu, Ala, Acc, Aib,Nle, Ile, Cha, Tle, Phe, X¹-Phe, Ala or deleted; A³³ is Val, Acc, Aib,Leu, Ile, Tle, Nle, Cha, Ala, Phe, Abu, X¹-Phe, Tba, Gaba or deleted;A³⁴ is Lys, Arg, hArg, Orn, Amp, Gaba, HN—CH((CH₂), —NR¹⁰R¹¹)—C(O),HN—CH((CH₂)_(e)—X³)—C(O) or deleted; A³⁵ is Gly or deleted; A³⁶ is L- orD-Arg, D- or L-Lys, D- or L-hArg, D- or L-Orn, Amp, HN—CH((CH₂),—NR¹⁰R¹¹)—C(O), HN—CH((CH₂)_(e)—X³)—C(O) or deleted; A³⁷ is Gly ordeleted; X¹ for each occurrence is independently selected from the groupconsisting of (C₁-C₆)alkyl, OH and halo; R¹ is OH, NH₂, (C₁-C₁₂)alkoxy,or NH—X²—CH₂-Z⁰, wherein X² is a (C₁-C₁₂)hydrocarbon moiety, and Z⁰ isH, OH, CO₂H or CONH₂; X³ is

or —C(O)—NHR¹², wherein X⁴ for each occurrence is independently —C(O)—,—NH—C(O)— or —CH₂—, and f for each occurrence is independently aninteger from 1 to 29; each of R² and R³ is independently selected fromthe group consisting of H, (C₁-C₃₀)alkyl, (C₂-C₃₀)alkenyl,phenyl(C₁-C₃₀)alkyl, naphthyl(C₁-C₃₀)alkyl, hydroxy(C₁-C₃₀)alkyl,hydroxy(C₂-C₃₀)alkenyl, hydroxyphenyl(C₁-C₃₀)alkyl, andhydroxynaphthyl(C₁-C₃₀)alkyl; or one of R² and R³ is C(O)X⁵ in which X⁵is (C₁-C₃₀)alkyl, (C₂-C₃₀)alkenyl, phenyl(C₁-C₃₀)alkyl,naphthyl(C₁-C₃₀)alkyl, hydroxy(C₁-C₃₀)alkyl, hydroxy(C₂-C₃₀)alkenyl,hydroxyphenyl(C₁-C₃₀)alkyl, hydroxynaphthyl(C₁-C₃₀)alkyl,

where Y is H or OH, r is 0 to 4 and q is 0 to 4; e for each occurrenceis independently an integer from 1 to 4; n for each occurrence isindependently an integer from 1-5; and R¹⁰ and R¹¹ for each occurrenceis each independently H, (C₁-C₃₀)alkyl, (C₁-C₃₀)acyl,(C₁-C₃₀)alkylsulfonyl, —C((NH)(NH₂)) or

provided that when R¹⁰ is (C₁-C₃₀)acyl, (C₁-C₃₀)alkylsulfonyl,—C((NH)(NH₂)) or

R¹¹ is H or (C₁-C₃₀)alkyl; and R¹² is (C₁-C₃₀)alkyl; with the provisothat: (i) at least one amino acid of a compound of formula (I) is notthe same as the native sequence of hGLP-1 (7-36, or -37)NH₂ (SEQ ID NOS:1, 2) or hGLP-1 (7-36, or -37)OH (SEQ ID NOS: 3, 4); (ii) a compound offormula (I) is not an analogue of hGLP-1(7-36, or -37)NH₂ (SEQ ID NOS:1,2) or hGLP-1(7-36, or -37)OH (SEQ ID NOS: 3, 4) wherein a singleposition has been substituted by Ala; (iii) a compound of formula (I) isnot [Lys²⁶(N^(ε)-alkanoyl)]hGLP-1(7-36, or -37)-E (SEQ ID NOS: 5-8),[Lys³⁴(N^(ε)-alkanoyl)]hGLP-1(7-36, or -37)-E (SEQ ID NOS: 9-12),[Lys^(26,34)-bis(N^(ε)-alkanoyl)]hGLP-1(7-36, or -37)-E (SEQ ID NOS:13-16), [Arg²⁶, Lys³⁴(NE-alkanoyl)]hGLP-1(8-36, or -37)-E (SEQ ID NOS:17-20), or [Arg^(26,34), Lys³⁶(N^(ε)-alkanoyl)]hGLP-1(7-36, or -37)-E,wherein E is —OH or —NH₂ (SEQ ID NOS: 21-24); (iv) a compound of formula(I) is not Z¹-hGLP-1(7-36, or -37)-OH, Z¹-hGLP-1(7-36, or -37)-NH₂,where Z¹ is selected from the group consisting of (a) [Arg²⁶] (SEQ IDNOS: 25-28), [Arg³⁴] (SEQ ID NOS: 29-32), [Arg^(26,34)] (SEQ ID NOS:33-36), [Lys³⁶] (SEQ ID NOS: 37-40), [Arg²⁶, Lys³⁶] (SEQ ID NOS: 41-44),[Arg³⁴, Lys³⁶] (SEQ ID NOS: 45-46), [D-Lys³⁶], [Arg³⁶] (SEQ ID NOS:37-40), [D-Arg³⁶], [Arg^(26,34), Lys³⁶] (SEQ ID NOS: 49-52), or[Arg^(26,36), Lys³⁴] (SEQ ID NOS: 25-28); (b) [Asp²¹] (SEQ ID NOS:53-56); (c) at least one of [Aib⁸] (SEQ ID NOS: 57-60), [D-Ala⁸] and[Asp⁹] (SEQ ID NOS: 61-64); and (d) [Tyr⁷] (SEQ ID NOS: 65-68),[N-acyl-His⁷] (SEQ ID NOS: 69-72), [N-alkyl-His⁷] (SEQ ID NOS: 73-76),[N-acyl-D-His⁷] or [N-alkyl-D-His⁷]; (v) a compound of formula (I) isnot a combination of any two of the substitutions listed in groups (a)to (d); and (vi) a compound of formula (I) is not [N-Me-Ala⁸]hGLP-1(8-36or -37) (SEQ ID NOS: 77, 78), [Glu¹⁵]hGLP-1(7-36 or -37) (SEQ ID NOS:79, 80), [Asp²¹]hGLP-1(7-36 or -37) (SEQ ID NOS: 53, 54) or[Phe³¹]hGLP-1(7-36 or -37) (SEQ ID NOS: 81, 82).
 2. A compound accordingto claim 1 or a pharmaceutically acceptable salt thereof wherein A¹¹ isThr; A¹³ is Thr; A¹⁴ is Ser, Aib or Ala; A¹⁷ is Ser, Ala, Aib or D-Ala;A¹⁸ is Ser, Ala, Aib or D-Ala; A²¹ is Glu or Ala; A²³ is Gln, Glu, orAla; and A²⁷ is Glu or Ala.
 3. A compound according to claim 2 or apharmaceutically acceptable salt thereof wherein A⁹ is Glu, N-Me-Glu orN-Me-Asp; A¹² is Phe, Acc or Aic; A¹⁶ is Val, D-Val, Acc, Aib, Ala, Tleor D-Ala; A¹⁹ is Tyr, 3-Pal, 4-Pal or D-Tyr; A²⁰ is Leu, Acc, Cha, Alaor Tle; A²⁴ is Ala, Aib or Acc; A²⁵ is Ala, Aib, Acc, Lys, Arg, hArg,Orn, HN—CH((CH₂), —NH—R¹⁰)—C(O); A²⁸ is Phe or Ala; A²⁹ is Ile, Acc orTle; A³⁰ is Ala, Aib or deleted; A³¹ is Trp, Ala, 3-Pal, 4-Pal ordeleted; A³² is Leu, Acc, Cha, Ala or deleted; A³³ is Val, Acc, Ala,Gaba, Tle or deleted.
 4. A compound according to claim 3 or apharmaceutically acceptable salt thereof wherein A⁸ is Ala, D-Ala, Aib,A6c, A5c, N-Me-Ala, N-Me-D-Ala or N-Me-Gly; A¹⁰ is Gly, Ala, D-Ala orPhe; A¹² is Phe, A6c or A5c; A¹⁶ is Val, Ala, Tle, A6c, A5c or D-Val;A²⁰ is Leu, A6c, A5c, Cha, Ala or Tle; A²² is Gly, Aib, β-Ala, L-Ala orD-Ala; A²⁴ is Ala or Aib; A²⁹ is Ile, A6c, A5c or Tle; A³² is Leu, A6c,A5c, Cha, Ala or deleted; A³³ is Val, A6c, A5c, Ala, Gaba, Tle ordeleted.
 5. A compound according to claim 4 or a pharmaceuticallyacceptable salt thereof wherein R¹ is OH or NH₂.
 6. A compound accordingto claim 5 or a pharmaceutically acceptable salt thereof wherein R² is Hand R³ is (C₁-C₃₀)alkyl, (C₂-C₃₀)alkenyl, (C₁-C₃₀)acyl,


7. A compound according to claim 1 wherein said compound is [D-Ala⁸,Ala^(17,22,23,27), 3-Pal^(19,31), Gaba³⁴]-GLP-1(7-34)NH₂;[D-Ala^(8,23,27), 3-Pal^(19,31)]hGLP-1(7-35)-NH₂; [Ala^(18,23,27),3-Pal^(19,31)]hGLP-1(7-35)-NH₂ (SEQ ID NO: 83); [Ala^(16,23,27),3-Pal^(19,31)]hGLP-1(7-35)-NH₂ (SEQ ID NO: 84); [Ala^(14,23,27),3-Pal^(19,31)]hGLP-1(7-35)-NH₂ (SEQ ID NO: 85); [Ala^(22,23,27),3-Pal^(19,31)]hGLP-1(7-35)-NH₂ (SEQ ID NO: 86); [Hppa⁷]hGLP-1(7-36)-NH₂(SEQ ID NO: 87); [Ala^(15,23,27), 3-Pal^(19,31)]hGLP-1(7-35)-NH₂ (SEQ IDNO: 88); [Ala^(17,23,27), 3-Pal^(19,31)]hGLP-1(7-35)-NH₂ (SEQ ID NO:89); [Ala^(22,23,27), 3-Pal^(19,31), Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO:90); [Ala^(15,23,27), 3-Pal^(19,31), Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO:91); [Ala^(17,23,27), 3-Pal^(19,31), Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO:92); [Ala^(18,22,23,27), 3-Pal^(19,31), Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ IDNO: 93); [Ala^(21,22,23,27), 3-Pal^(19,31), Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQID NO: 94); [Ala^(22,23,26,27), 3-Pal^(19,31), Gaba³⁴]hGLP-1(7-34)-NH₂(SEQ ID NO: 95); [Ala^(22,23,27,32), 3-Pal^(19,31),Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 96); [Ala^(22,23,26,27),3-Pal^(19,31), Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 97);[Ala^(22,23,27,31), 3-Pal¹⁹, Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 98);[Ala^(22,23,27,28), 3-Pal^(19,31), Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO:99); [Ala^(22,23,27,29), 3-Pal^(19,31), Gaba³³]hGLP-1(7-33)-NH₂ (SEQ IDNO: 100); [Ala^(23,27), 3-Pal^(19,31), Gaba³³]hGLP-1(7-33)-NH₂ (SEQ IDNO: 101); [Ala^(20,22,23,27), 3-Pal^(19,31), Gaba³³]hGLP-1(7-33)-NH₂(SEQ ID NO: 102); [Ala^(22,23,27), 3-Pal^(19,31),Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 103); [Ala^(17,22,23,27),3-Pal^(19,31), Gaba³³]hGLP-1(7-33)-NH₂ (SEQ ID NO: 104); [D-Ala¹⁰,Ala^(22,23,27), 3-Pal^(19,31), Gaba³³]hGLP-1(7-33)-NH₂; [D-Ala⁸,Ala^(17,23,27), 3-Pal^(19,31)]hGLP-1(7-34)-NH₂; [Ala^(17,23,27),3-Pal^(19,26,31)]hGLP-1(7-34)-NH₂ (SEQ ID NO: 105); [D-Ala⁸, Ala¹⁷,3-Pal^(19,31)]hGLP-1(7-34)-NH₂; [Ala^(17,23,27),3-Pal^(19,31)]hGLP-1(7-34)-NH₂ (SEQ ID NO: 106); [D-Ala⁸,Ala^(17,23,27), 3-Pal^(19,31), Tle²⁹]hGLP-1(7-34)-NH₂; [D-Ala⁸,Ala^(17,23,27), 3-Pal^(19,31), Tle¹⁶]hGLP-1(7-34)-NH₂; [D Ala⁸,Ala^(17,23,27), 3-Pal^(19,31), Gaba³⁴]hGLP-1(7-34)-NH₂; [D-Ala²²,Ala^(17,23,27), 3-Pal^(19,31), Gaba³⁴]hGLP-1(7-34)-NH₂; [Aib⁸,Ala^(17,23,27), 3-Pal^(19,31), Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 107);[D-Ala⁸, Ala^(17,22,23,27), 3-Pal^(19,31)]hGLP-1(7-33)-NH₂; [Aib⁸,Ala^(17,22,23,27), 3-Pal^(19,31)]hGLP-1(7-33)-NH₂ (SEQ ID NO: 108);[Ala^(17,18,23,27), 3-Pal^(19,31), Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO:109); [Ala^(17,23,27), 3-Pal^(19,31), Tle³³, Gaba³⁴]hGLP-1(7-34)-NH₂(SEQ ID NO: 110); [Tle¹⁶, Ala^(17,23,27), 3-Pal^(19,31),Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 111); [N-Me-D-Ala⁸,Ala^(17,22,23,27), 3-Pal^(19,31)]hGLP-1(7-33)-NH₂; [Aib⁸,Ala^(17,18,23,27), 3-Pal^(19,31)]hGLP-1(7-33)-NH₂ (SEQ ID NO: 112);[Ala^(17,18,22,23,27), 3-Pal^(19,31), Tle^(16,20),Gaba³⁴]hGLP-1(7-34)-NH₂ (SEQ ID NO: 113); [D-Ala⁸, Ala^(17,18,22,23,27),3-Pal^(19,31), Tle¹⁶, Gaba³⁴]hGLP-1(7-34)-NH₂; [D-Ala^(8,22),Ala^(17,18,23,27,3)-Pal^(19,31), Gaba³⁴]hGLP-1(7-34)-NH₂; [D-Ala^(8,18),Ala^(17,22,23,27), 3-Pal^(19,31), Gaba³⁴]hGLP-1(7-34)-NH₂;[D-Ala^(8,17), Ala^(18,22,23,27), 3-Pal^(19,31),Gaba³⁴]hGLP-1(7-34)-NH₂; or [D-Ala⁸, Ala^(17,18,22,23,27),3-Pal^(19,31), Gaba³⁴]hGLP-1(7-34)-NH₂; or a pharmaceutically acceptablesalt thereof.
 8. A compound according to claim 1 wherein said compoundis [Aib⁸, A6c³²]hGLP-1(7-36)NH₂ (SEQ ID NO: 114);[A6c^(20,32)]hGLP-1(7-36)-NH₂ (SEQ ID NO: 115); [Aib⁸]hGLP-1(7-36)-NH₂(SEQ ID NO: 116); [(Tma-His)⁷]hGLP-1(7-36)-NH₂ (SEQ ID NO: 117);[A6c⁸]hGLP-1(8-36)-NH₂ (SEQ ID NO: 118); [A6c⁸]hGLP-1(7-36)-NH₂ (SEQ IDNO: 119); [A6c^(16,20)]hGLP-1(7-36)-NH₂ (SEQ ID NO: 120);[A6c^(29,32)]hGLP-1(7-36)-NH₂ (SEQ ID NO: 121); [A6c²⁰,Aib²⁴]hGLP-1(7-36)-NH₂ (SEQ ID NO: 122); [Aib²⁴,A6c^(29,32)]hGLP-1(7-36)-NH₂ (SEQ ID NO: 123);[A6c^(16,29,32)]hGLP-1(7-36)-NH₂ (SEQ ID NO: 124);[Ura⁷]hGLP-1(7-36)-NH₂ (SEQ ID NO: 125); [Paa⁷]hGLP-1(7-36)-NH₂ (SEQ IDNO: 126); [Pta⁷]hGLP-1(7-36)-NH₂ (SEQ ID NO: 127);[N-Me-Ala⁸]hGLP-1(7-36)-NH₂ (SEQ ID NO: 128);[N-Me-Ala⁸]hGLP-1(8-36)-NH₂ (SEQ ID NO: ______);[N-Me-D-Ala⁸]hGLP-1(7-36)-NH₂; [N-Me-D-Ala⁸]hGLP-1(8-36)-NH₂;[N-Me-Gly⁸]hGLP-1(7-36)-NH₂ (SEQ ID NO: 129); [A5c⁸]hGLP-1(7-36)-NH₂(SEQ ID NO: 130); [N-Me-Glu⁹]hGLP-1(7-36)-NH₂ (SEQ ID NO: 131); [A5c⁸,A6c^(20,32)]hGLP-1(7-36)-NH₂ (SEQ ID NO: 132); [Aib⁸,A6c³²]hGLP-1(7-36)-NH₂ (SEQ ID NO: 133); [Aib^(8,25)]hGLP-1(7-36)-NH₂(SEQ ID NO: 134); [Aib⁸²⁴]hGLP-1(7-36)-NH₂ (SEQ ID NO: 135);[Aib^(8.30)]hGLP-1(7-36)-NH₂ (SEQ ID NO: 136); [Aib⁸,Cha²⁰]hGLP-1(7-36)-NH₂ (SEQ ID NO: 137); [Aib⁸, Cha³²]hGLP-1(7-36)-NH₂(SEQ ID NO: 138); [Aib⁸, Glu²³]hGLP-1(7-36)-NH₂ (SEQ ID NO: 139); [Aib⁸,A6c²⁰]hGLP-1(7-36)-NH₂ (SEQ ID NO: 140); [Aib⁸,A6c^(20.32)]hGLP-1(7-36)-NH₂ (SEQ ID NO: 141);[Aib^(8,22)]hGLP-1(7-36)-NH₂ (SEQ ID NO: 142); [Aib⁸,β-Ala²²]hGLP-1(7-36)-NH₂ (SEQ ID NO: 143); [Aib⁸, Lys²⁵]hGLP-1(7-36)-NH₂(SEQ ID NO: 144); [Aib⁸, A6c¹²]hGLP-1(7-36)-NH₂ (SEQ ID NO: 145); [Aib⁸,A6c²⁹]hGLP-1(7-36)-NH₂ (SEQ ID NO: 146); [Aib⁸, A6c³³]hGLP-1(7-36)-NH₂(SEQ ID NO: 147); [Aib^(8,14)]hGLP-1(7-36)NH₂ (SEQ ID NO: 148);[Aib^(8,18)]hGLP-1(7-36)NH₂ (SEQ ID NO: 149);[Aib^(8,17)]hGLP-1(7-36)NH₂ (SEQ ID NO: 150); or [Aib⁸,D-Arg;²⁶]hGLP-1(7-36)NH₂; or a pharmaceutically acceptable salt thereof.9. A pharmaceutical composition comprising an effective amount of acompound according to claim 1 or a pharmaceutically acceptable saltthereof and a pharmaceutically acceptable carrier or diluent.
 10. Amethod of eliciting an agonist effect from a GLP-1 receptor in a subjectin need thereof which comprises administering to said subject aneffective amount of a compound according to claim 1 or apharmaceutically acceptable salt thereof.
 11. A method of treating adisease selected from the group consisting of Type I diabetes, Type IIdiabetes, obesity, glucagonomas, secretory disorders of the airway,metabolic disorder, arthritis, osteoporosis, central nervous systemdisease, restenosis, neurodegenerative disease, renal failure,congestive heart failure, nephrotic syndrome, cirrhosis, pulmonaryedema, and hypertension, in a subject in need thereof which comprisesadministering to said subject an effective amount of a compoundaccording to claim 1 or a pharmaceutically acceptable salt thereof. 12.A method according to claim 11 wherein said disease is Type I diabetesor Type II diabetes.